Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein, HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease, Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. Whether these are direct or indirect effects of HFE function is not known. In order to address this question and to understand the molecular mechanisms by which HFE influences iron homeostasis, cell lines have been developed that when transfected with HFE show either increases or decreases in iron levels. The aims of this proposal are focused on finding the basis of the opposite effects of HFE function in different cell types. The hypothesis that HFE alters iron homeostasis by interacting different proteins in different cell lines will be tested. Chimeric HFE proteins will be made to define the domains of HFE responsible for the alterations in iron homeostasis and site-directed mutagenesis will be used for finer mapping of the domains. The HFE protein complex could be directly regulating intracellular iron levels or signaling independently of intracellular iron levels. New genes that functionally couple to HFE will be identified in a high throughput screen. The hypothesis that HFE alters gene expression in an iron-dependent manner will be tested by microarray analysis of cells expressing and not expressing HFE. Finally, the hypothesis that HFE acts to regulate iron homeostasis in hepatocytes solely through controlling intracellular iron levels will be tested using a hepatic cell line and orimary hepatocytes. The long term goal of this work is to understand the function of HFE.